The organisms found in the microbiome outnumber human cells significantly. This makes the microbiome a key target to aid in our understanding of health and disease. In this article, we summarise a recent webinar talk by Caroline Wensel (PhD Candidate, Sears Lab,Johns Hopkins School of Medicine), which explores why we should sequence the human microbiome, how we can do so, and the associated challenges.
Why should we sequence the microbiome?
Microbial cells vastly outnumber human cells. With such a wide array of lifeforms finding homes in our bodies – including bacteria, fungi and viruses – it is vital that we understand the influence this has on our health and wellbeing.
Most of these microbes are harmless or beneficial. Yet, many can be pathogenic. A range diseases suffered by humans are attributed in part to microbial dysbiosis – when the composition of the microbiome is altered from its typical state. These diseases include, but are not limited to, inflammatory bowel disease, diabetes, obesity and even cancer. Understanding the composition of the microbiome and its contribution to these conditions requires us to have an intimate knowledge of these microbes. But this isn’t always easy.
Some bacteria are difficult to culture using traditional methods, and some cannot be cultured using these methods at all. Plus, culturing multiple samples can be costly and time-consuming. In addition, the presence of functional redundancy dictates the need for better understanding of bacterial function in the microbiome. These problems can be solved using sequencing – but this is not always a simple task.
How can we sequence the microbiome?
Common methods to sequence the microbiome include amplicon sequencing, shotgun metagenomics and RNA sequencing. Key differences between these techniques are the genetic material that is sequenced, and the resolution and coverage at which they can differentiate between microbial species.
The cost of these techniques is directly correlated to the amount of information you can obtain through their use. Amplicon sequencing is by far the cheapest option, but can only be used to differentiate between bacteria, archaea and eukaryotes. It cannot be used for direct functional profiling, but makes up for these shortcomings with a lower false positive rate. Conversely, RNA sequencing can be used for active functional profiling and can differentiate between individual microbial strains – including that of viruses.
Ultimately, each method presents its own benefits and challenges – the best method for you will depend on your research question.
Challenges of rigor, reproducibility and reporting
The microbiome varies vastly between individuals. This is influenced by age, sex and environmental factors such as geographic location, diet and medications. Plus, an individual’s own genetics and epigenetics can influence the makeup of their microbiome. In addition, experimental setup, sample type, sequencing method and other factors can influence the variability of results, even in samples from the same individual.
To complicate matters further, analytical choices can also influence microbiome experiments. Differences in analysis pipelines can provide different results, as can other important steps such as contaminant removal and statistical modelling. Recently, high profile retractions have highlighted the need for better standards when it comes to reporting results from microbiome studies, in a bid to overcome these problems.
What is the takeaway? Caroline says, ‘There is no one correct approach. But it’s really important to treat all the samples the same in your study to facilitate meaningful comparisons, and then report anything you did.’
Read Caroline’s review on next-generation sequencing of the microbiome here.
Watch the full webinar on demand here.
